Mycobacterium tuberculosis (Mtb) infects one third of the world's population and kills approximately 2 million people per year worldwide. Despite over a century of investigation, the molecular mechanisms of Mtb pathogenesis are still largely unknown. We are investigating the role of the Mtb cell envelope in pathogenesis through the creation and characterization Mtb mutants. Through this approach, we have established that two members of a novel gene family in Mtb, the mycolic acid cyclopropane synthetases, modify the major lipids of the Mtb cell envelope with specific stereochemistries of cyclopropyl residues. In addition, we have established that deletion of individual cyclopropane synthetases has dramatic effects on the pathogenesis of Mtb infection in mice, including alterations in the host immune response and consequent tissue destruction. To further study the role of mycolic acid cyclopropanation in Mtb pathogenesis, we have generated a set of cyclopropane synthetase mutants through allelic exchange. We will define the function of each these genes in mycolic acid modification through the examination of mycolic acid profiles in the mutant strains. Furthermore, we will define the pathogenetic role of each synthetase in mouse models of Mtb infection. We will study the mechanism by which cyclopropanated mycolic acids affect pathogenesis by characterizing the Trehalose Dimycolate (TDM) from the mutant strains and examining the role of innate immunity in the recognition of cyclopropane deficient mycolic acids. These studies will elucidate the role of mycolic acid cyclopropane modification in Mtb pathogenesis and examine the mechanism by which the fine chemical structure of the Mtb mycolic acids mediates symbiosis in vivo. These studies will contribute to our long-term goal of understanding the relationship between the chemical complexity of the Mtb cell envelope and the pathogenesis of Mtb infection. This knowledge may validate this new antibiotic target in Mtb and lead to new vaccine strategies.